A Novel Heterozygous Mutation c.1627G>T (p.Gly543Cys) in the SLC34A1 Gene in a Male Patient with Recurrent Nephrolithiasis and Early Onset Osteopenia: A Case Report.

Serum phosphate concentration is regulated by renal phosphate reabsorption and mediated by sodium-phosphate cotransporters. Germline mutations in genes encoding these cotransporters have been associated with clinical phenotypes, variably characterized by hyperphosphaturia, hypophosphatemia, recurrent kidney stones, skeletal demineralization, and early onset osteoporosis. We reported a 33-year-old male patient presenting a history of recurrent nephrolithiasis and early onset osteopenia in the lumbar spine and femur. He was tested, through next generation sequencing (NGS), by using a customized multigenic panel containing 33 genes, whose mutations are known to be responsible for the development of congenital parathyroid diseases. Two further genes, SLC34A1 and SLC34A3, encoding two sodium-phosphate cotransporters, were additionally tested. A novel germline heterozygous mutation was identified in the SLC34A1 gene, c.1627G>T (p.Gly543Cys), currently not reported in databases of human gene mutations and scientific literature. SLC34A1 germline heterozygous mutations have been associated with the autosomal dominant hypophosphatemic nephrolithiasis/osteoporosis type 1 (NPHLOP1). Consistently, alongside the clinical features of NPHLOP1, our patient experienced recurrent nephrolithiasis and lumbar and femoral osteopenia at a young age. Genetic screening for the p.Gly453Cys variant and the clinical characterization of his first-degree relatives associated the presence of the variant in one younger brother, presenting renal colic and microlithiasis, suggesting p.Gly453Cys is possibly associated with renal altered function in the NPHLOP1 phenotype.

Understanding renal phosphate handling: unfinished business.

PURPOSE OF REVIEW: The purpose of this review is to highlight the publications from the prior 12-18 months that have contributed significant advances in the field of renal phosphate handling. RECENT FINDINGS: The discoveries include new mechanisms for the trafficking and expression of the sodium phosphate cotransporters; direct link between phosphate uptake and intracellular metabolic pathways; interdependence between proximal tubule transporters; and the persistent renal expression of phosphate transporters in chronic kidney disease. SUMMARY: Discovery of new mechanisms for trafficking and regulation of expression of phosphate transporters suggest new targets for the therapy of disorders of phosphate homeostasis. Demonstration of stimulation of glycolysis by phosphate transported into a proximal tubule cell expands the scope of function for the type IIa sodium phosphate transporter from merely a mechanism to reclaim filtered phosphate to a regulator of cell metabolism. This observation opens the door to new therapies for preserving kidney function through alteration in transport. The evidence for persistence of active renal phosphate transport even with chronic kidney disease upends our assumptions of how expression of these transporters is regulated, suggests the possibility of alternative functions for the transporters, and raises the possibility of new therapies for phosphate retention.

FGF23 functions and disease.

The main function of fibroblast growth factor 23 (FGF23) is the regulation of phosphate metabolism through its action on the sodium-dependent phosphate cotransporters in the proximal renal tubules. Additionally, FGF23 interacts with vitamin D and parathyroid hormone in a complex metabolic pathway whose detailed mechanisms are still not clear in human physiology and disease. More recently, research has also focused on the understanding of mechanisms of FGF23 action on organs and system other than the kidneys and bone, as well as on its interaction with other metabolic pathways. Collectively, the new evidence are successfully used for the clinical evaluation and management of FGF23-related disorders, for which new therapies with many potential applications are now available.

Analysis of sex difference in the tubular reabsorption of lithium in rats.

Lithium is used in the treatment of bipolar disorder. We previously demonstrated that two types of transporters mediate the tubular reabsorption of lithium in rats, and suggested that sodium-dependent phosphate transporters play a role in lithium reabsorption with high affinity. In the present study, we examined sex differences in lithium reabsorption in rats. When lithium chloride was infused at 60 µg/min, creatinine clearance and the renal clearance of lithium were lower, and the plasma concentration of lithium was higher in female rats. These values reflected the higher fractional reabsorption of lithium in female rats. In rats infused with lithium chloride at 6 µg/min, the pharmacokinetic parameters of lithium examined were all similar in both sexes. The fractional reabsorption of lithium was decreased by foscarnet, a representative inhibitor of sodium-dependent phosphate transporters, in male and female rats when lithium chloride was infused at the low rate. Among the candidate transporters mediating lithium reabsorption examined herein, the mRNA expression of only PiT2, a sodium-dependent phosphate transporter, exhibited sexual dimorphism. The present results demonstrated sex differences in the tubular reabsorption of lithium with low affinity in rats.

Novel mineral regulatory pathways in ovine pregnancy: I. phosphate, klotho signaling, and sodium-dependent phosphate transporters.

Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.

Modulation of Intestinal Phosphate Transport in Young Goats Fed a Low Phosphorus Diet.

The intestinal absorption of phosphate (Pi) takes place transcellularly through the active NaPi-cotransporters type IIb (NaPiIIb) and III (PiT1 and PiT2) and paracellularly by diffusion through tight junction (TJ) proteins. The localisation along the intestines and the regulation of Pi absorption differ between species and are not fully understood. It is known that 1,25-dihydroxy-vitamin D3 (1,25-(OH)2D3) and phosphorus (P) depletion modulate intestinal Pi absorption in vertebrates in different ways. In addition to the apical uptake into the enterocytes, there are uncertainties regarding the basolateral excretion of Pi. Functional ex vivo experiments in Ussing chambers and molecular studies of small intestinal epithelia were carried out on P-deficient goats in order to elucidate the transepithelial Pi route in the intestine as well as the underlying mechanisms of its regulation and the proteins, which may be involved. The dietary P reduction had no effect on the duodenal and ileal Pi transport rate in growing goats. The ileal PiT1 and PiT2 mRNA expressions increased significantly, while the ileal PiT1 protein expression, the mid jejunal claudin-2 mRNA expression and the serum 1,25-(OH)2D3 levels were significantly reduced. These results advance the state of knowledge concerning the complex mechanisms of the Pi homeostasis in vertebrates.

Phosphate Transport in Epithelial and Nonepithelial Tissue.

Phosphate is an essential nutrient for life and is a critical component of bone formation, a major signaling molecule, and structural component of cell walls. Phosphate is also a component of high-energy compounds (i.e., AMP, ADP, and ATP) and essential for nucleic acid helical structure (i.e., RNA and DNA). Phosphate plays a central role in the process of mineralization, normal serum levels being associated with appropriate bone mineralization, while high and low serum levels are associated with soft tissue calcification. The serum concentration of phosphate and the total body content of phosphate are highly regulated, a process that is accomplished by the coordinated effort of two families of sodium-dependent transporter proteins. The three isoforms of the SLC34 family (SLC34A1-A3) show very restricted tissue expression and regulate intestinal absorption and renal excretion of phosphate. SLC34A2 also regulates the phosphate concentration in multiple lumen fluids including milk, saliva, pancreatic fluid, and surfactant. Both isoforms of the SLC20 family exhibit ubiquitous expression (with some variation as to which one or both are expressed), are regulated by ambient phosphate, and likely serve the phosphate needs of the individual cell. These proteins exhibit similarities to phosphate transporters in nonmammalian organisms. The proteins are nonredundant as mutations in each yield unique clinical presentations. Further research is essential to understand the function, regulation, and coordination of the various phosphate transporters, both the ones described in this review and the phosphate transporters involved in intracellular transport.

A single Na+-Pi cotransporter in Toxoplasma plays key roles in phosphate import and control of parasite osmoregulation.

Inorganic ions such as phosphate, are essential nutrients required for a broad spectrum of cellular functions and regulation. During infection, pathogens must obtain inorganic phosphate (Pi) from the host. Despite the essentiality of phosphate for all forms of life, how the intracellular parasite Toxoplasma gondii acquires Pi from the host cell is still unknown. In this study, we demonstrated that Toxoplasma actively internalizes exogenous Pi by exploiting a gradient of Na+ ions to drive Pi uptake across the plasma membrane. The Na+-dependent phosphate transport mechanism is electrogenic and functionally coupled to a cipargarmin sensitive Na+-H+-ATPase. Toxoplasma expresses one transmembrane Pi transporter harboring PHO4 binding domains that typify the PiT Family. This transporter named TgPiT, localizes to the plasma membrane, the inward buds of the endosomal organelles termed VAC, and many cytoplasmic vesicles. Upon Pi limitation in the medium, TgPiT is more abundant at the plasma membrane. We genetically ablated the PiT gene, and ΔTgPiT parasites are impaired in importing Pi and synthesizing polyphosphates. Interestingly, ΔTgPiT parasites accumulate 4-times more acidocalcisomes, storage organelles for phosphate molecules, as compared to parental parasites. In addition, these mutants have a reduced cell volume, enlarged VAC organelles, defects in calcium storage and a slightly alkaline pH. Overall, these mutants exhibit severe growth defects and have reduced acute virulence in mice. In survival mode, ΔTgPiT parasites upregulate several genes, including those encoding enzymes that cleave or transfer phosphate groups from phosphometabolites, transporters and ions exchangers localized to VAC or acidocalcisomes. Taken together, these findings point to a critical role of TgPiT for Pi supply for Toxoplasma and also for protection against osmotic stresses.

Isolation and Characterization of Three Sodium-Phosphate Cotransporter Genes and Their Transcriptional Regulation in the Grass Carp Ctenopharyngodon idella.

It is important to explore the regulatory mechanism of phosphorus homeostasis in fish, which help avoid the risk of P toxicity and prevent P pollution in aquatic environment. The present study obtained the full-length cDNA sequences and the promoters of three SLC20 members (slc20a1a, slc20a1b and slc20a2) from grass carp Ctenopharyngodon idella, and explored their responses to inorganic phosphorus (Pi). Grass carp SLC20s proteins possessed conservative domains and amino acid sites relevant with phosphorus transport. The mRNAs of three slc20s appeared in the nine tissues, but their expression levels were tissue-dependent. The binding sites of three transcription factors (SREBP1, NRF2 and VDR) were predicted on the slc20s promoters. The mutation and EMSA analysis indicated that: (1) SREBP1 binding site (-783/-771 bp) negatively but VDR (-260/-253 bp) binding site positively regulated the activities of slc20a1a promoter; (2) SREBP1 (-1187/-1178 bp), NRF2 (-572/-561 bp) and VDR(615/-609 bp) binding sites positively regulated the activities of slc20a1b promoter; (3) SREBP1 (-987/-977 bp), NRF2 (-1469/-1459 bp) and VDR (-1124/-1117 bp) binding sites positively regulated the activities of the slc20a2 promoter. Moreover, Pi incubation significantly reduced the activities of three slc20s promoters, and Pi-induced transcriptional inactivation of slc20s promoters abolished after the mutation of the VDR element but not SREBP1 and NRF2 elements. Pi incubation down-regulated the mRNA levels of three slc20s. For the first time, our study elucidated the transcriptional regulatory mechanisms of SLC20s and their responses to Pi, which offered new insights into the Pi homeostatic regulation and provided the basis for reducing phosphorus discharge into the waters.

Potential interaction of inflammatory hyperemia and hyperphosphatemia in tumorigenesis.

The present article proposes that the association of inflammation with cancer is potentially mediated by the interaction of inflammatory hyperemia and hyperphosphatemia. Hyperemia increases blood flow rate and blood volume, and hyperphosphatemia is caused by elevated serum levels of dysregulated inorganic phosphate. It is hypothesized that the interaction of inflammatory hyperemia and hyperphosphatemia circulates increased amounts of inorganic phosphate to the tumor microenvironment, where increased uptake of inorganic phosphate through sodium-phosphate cotransporters is sequestered in cells. Elevated levels of intracellular phosphorus increase biosynthesis of ribosomal RNA, leading to increased protein synthesis that supports tumor growth. The present article also proposes that the interaction of inflammatory hyperemia and hyperphosphatemia may help explain a chemopreventive mechanism associated with NSAIDs.

Tissue-Wide Gene Expression Analysis of Sodium/Phosphate Co-Transporters in Pigs.

Sodium/phosphate co-transporters are considered to be important mediators of phosphorus (P) homeostasis. The expression of specific sodium/phosphate co-transporters is routinely used as an immediate response to dietary interventions in different species. However, a general understanding of their tissue-specificity is required to elucidate their particular contribution to P homeostasis. In this study, the tissue-wide gene expression status of all currently annotated sodium/phosphate co-transporters were investigated in two pig trials focusing on a standard commercial diet (trial 1) or divergent P-containing diets (trial 2). A wide range of tissues including the gastrointestinal tract (stomach, duodenum, jejunum, ileum, caecum, and colon), kidney, liver, bone, muscle, lung, and aorta were analyzed. Both trials showed consistent patterns in the overall tissue-specific expression of P transporters. While SLC34A2 was considered as the most important intestinal P transporter in other species including humans, SLC34A3 appeared to be the most prominent intestinal P transporter in pigs. In addition, the P transporters of the SLC17 family showed basal expression in the pig intestine and might have a contribution to P homeostasis. The expression patterns observed in the distal colon provide evidence that the large intestine may also be relevant for intestinal P absorption. A low dietary P supply induced higher expressions of SLC20A1, SLC20A2, SLC34A1, and SLC34A3 in the kidney cortex. The results suggest that the expression of genes encoding transcellular P transporters is tissue-specific and responsive to dietary P supply, while underlying regulatory mechanisms require further analyses.

Suppressive effects of N-bisphosphonate in osteoblastic cells mitigated by non-N-bisphosphonate but not by sodium-dependent phosphate cotransporter inhibitor.

There are two types of bisphosphonates (BPs), nitrogen-containing (N-BPs) and those free from nitrogen (non-N-BPs). Although N-BPs show greater inhibition of bone resorption than non-N-BPs, their effects are likely accompanied with inflammation, which non-N-BPs mitigate. We examined the competitive effects of zoledronate (ZOL), an N-BP, and etidronate (ETI), a non-N-BP, in osteoblasts. ZOL, but not ETI, markedly reduced alkaline phosphatase activity and cell viability in osteoblastic MC3T3-E1 and Saos2 cells, while that inhibition was relieved by simultaneous administration of ETI, possibly because of competition with ZOL for cellular uptake. However, phosphonoformate, an inhibitor of the phosphonate transporters SLC20A and SLC34A, did not mitigate the reducing effects of ZOL, suggesting that those transporters are not involved in BP uptake in osteoblastic cells. Additionally, ZOL reduced fibroblastic NIH3T3 and C3H10T1/2 cell viability, which was relieved by administration of both ETI and phosphonoformate. Transporter gene expression levels were significantly lower in osteoblasts as compared with fibroblasts, which may account for the distinct effects of phosphonoformate with different cell types. Together, our results suggest existence of a common uptake route of N-BPs and non-N-BPs into osteoblastic cells that is unrelated to the SLC20A and SLC34A families. SIGNIFICANCE OF THE STUDY: N-BP ZOL was shown to suppress differentiation and viability of osteoblasts. ZOL-induced cell viability suppression was also observed in fibroblasts, which was markedly relieved by addition of the non-N-BP ETI. Additionally, mitigation of the effects of ZOL was achieved with phosphonoformate, a sodium-phosphate cotransporter inhibitor, in fibroblastic cells but not osteoblasts. Expression levels of SLC20A and SLC34A family genes were significantly lower in osteoblasts as compared with fibroblasts. These observations suggest that incorporation of N-BPs and non-N-BPs in osteoblasts is mediated via common transporters that appear to be distinct from SLC20A and 34A, which operate in fibroblasts.

Pyrophosphate Stimulates the Phosphate-Sodium Symporter of Trypanosoma brucei Acidocalcisomes and Saccharomyces cerevisiae Vacuoles.

Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes.IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

The role of sodium phosphate cotransporters in ectopic calcification.

Phosphate plays a critical role in many vital cellular processes. Deviations from normal serum phosphate levels, including alterations in the extracellular phosphate/pyrophosphate ratio, can cause severe consequences, such as ectopic calcification. Cellular phosphate levels are tightly controlled by sodium phosphate cotransporters, underscoring their importance in cellular physiology. The role of sodium phosphate cotransporters in ectopic calcification requires further elucidation, taking into account their important role in the control of intracellular phosphate levels and the synthesis of ATP, the main source of extracellular pyrophosphate (a potent endogenous inhibitor of calcification). In this review, we discuss the roles of phosphate and pyrophosphate homeostasis in ectopic calcification, with a specific focus on phosphate transporters. We concentrate on the five known sodium-dependent phosphate transporters and review their localisation and regulation by external factors, and the effects observed in knockout studies and in naturally occurring mutations.

Renal phosphate handling and inherited disorders of phosphate reabsorption: an update.

Renal phosphate handling critically determines plasma phosphate and whole body phosphate levels. Filtered phosphate is mostly reabsorbed by Na+-dependent phosphate transporters located in the brush border membrane of the proximal tubule: NaPi-IIa (SLC34A1), NaPi-IIc (SLC34A3), and Pit-2 (SLC20A2). Here we review new evidence for the role and relevance of these transporters in inherited disorders of renal phosphate handling. The importance of NaPi-IIa and NaPi-IIc for renal phosphate reabsorption and mineral homeostasis has been highlighted by the identification of mutations in these transporters in a subset of patients with infantile idiopathic hypercalcemia and patients with hereditary hypophosphatemic rickets with hypercalciuria. Both diseases are characterized by disturbed calcium homeostasis secondary to elevated 1,25-(OH)2 vitamin D3 as a consequence of hypophosphatemia. In vitro analysis of mutated NaPi-IIa or NaPi-IIc transporters suggests defective trafficking underlying disease in most cases. Monoallelic pathogenic mutations in both SLC34A1 and SLC34A3 appear to be very frequent in the general population and have been associated with kidney stones. Consistent with these findings, results from genome-wide association studies indicate that variants in SLC34A1 are associated with a higher risk to develop kidney stones and chronic kidney disease, but underlying mechanisms have not been addressed to date.

Proximal renal tubular acidosis with and without Fanconi syndrome

Proximal renal tubular acidosis (RTA) is caused by a defect in bicarbonate (HCO₃⁻) reabsorption in the kidney proximal convoluted tubule. It usually manifests as normal anion-gap metabolic acidosis due to HCO₃⁻ wastage. In a normal kidney, the thick ascending limb of Henle’s loop and more distal nephron segments reclaim all of the HCO₃⁻ not absorbed by the proximal tubule. Bicarbonate wastage seen in type II RTA indicates that the proximal tubular defect is severe enough to overwhelm the capacity for HCO₃⁻ reabsorption beyond the proximal tubule. Proximal RTA can occur as an isolated syndrome or with other impairments in proximal tubular functions under the spectrum of Fanconi syndrome. Fanconi syndrome, which is characterized by a defect in proximal tubular reabsorption of glucose, amino acids, uric acid, phosphate, and HCO₃⁻, can occur due to inherited or acquired causes. Primary inherited Fanconi syndrome is caused by a mutation in the sodium-phosphate cotransporter (NaPₐ-II) in the proximal tubule. Recent studies have identified new causes of Fanconi syndrome due to mutations in the EHHADH and the HNF4A genes. Fanconi syndrome can also be one of many manifestations of various inherited systemic diseases, such as cystinosis. Many of the acquired causes of Fanconi syndrome with or without proximal RTA are drug-induced, with the list of causative agents increasing as newer drugs are introduced for clinical use, mainly in the oncology field.

Infantile hypercalcemia with novel compound heterozygous mutation in SLC34A1 encoding renal sodium-phosphate cotransporter 2a: a case report

Idiopathic infantile hypercalcemia is characterized by hypercalcemia, dehydration, vomiting, and failure to thrive, and it is due to mutations in 24-hydroxylase (CYP24A1). Recently, mutations in sodium-phosphate cotransporter (SLC34A1) expressed in the kidney were discovered as an additional cause of idiopathic infantile hypercalcemia. This report describes a female infant admitted for evaluation of nephrocalcinosis. She presented with hypercalcemia, hypercalciuria, low intact parathyroid hormone level, and high 1,25-dihydroxyvitamin D3 level. Exome sequencing identified novel compound heterozygous mutations in SLC34A1 (c.1337G>A, c.1483C>T). The patient was treated with fluids for hydration, furosemide, a corticosteroid, and restriction of calcium/vitamin D intake. At the age of 7 months, the patient's calcium level was within the normal range, and hypercalciuria waxed and waned. Renal echogenicity improved on the follow-up ultrasonogram, and developmental delay was not noted. In cases of hypercalcemia with subsequent hypercalciuria, DNA analysis for SLC34A1 gene mutations and CYP24A1 gene mutations should be performed. Further studies are required to obtain long-term data on hypercalciuria and nephrocalcinosis.

5-Diphosphoinositol pentakisphosphate (5-IP7) regulates phosphate release from acidocalcisomes and yeast vacuoles.

Acidocalcisomes of Trypanosoma brucei and the acidocalcisome-like vacuoles of Saccharomyces cerevisiae are acidic calcium compartments that store polyphosphate (polyP). Both organelles possess a phosphate-sodium symporter (TbPho91 and Pho91p in T. brucei and yeast, respectively), but the roles of these transporters in growth and orthophosphate (Pi) transport are unclear. We found here that Tbpho91-/- trypanosomes have a lower growth rate under phosphate starvation and contain larger acidocalcisomes that have increased Pi content. Heterologous expression of TbPHO91 in Xenopus oocytes followed by two-electrode voltage clamp recordings disclosed that myo-inositol polyphosphates stimulate both sodium-dependent depolarization of the oocyte membrane potential and Pi conductance. Deletion of the SPX domain in TbPho91 abolished this stimulation. Inositol pyrophosphates such as 5-diphosphoinositol pentakisphosphate generated outward currents in Na+/Pi-loaded giant vacuoles prepared from WT or from TbPHO91-expressing pho91Δ strains but not from the pho91Δ yeast strains or from the pho91Δ strains expressing PHO91 or TbPHO91 with mutated SPX domains. Our results indicate that TbPho91 and Pho91p are responsible for vacuolar Pi and Na+ efflux and that myo-inositol polyphosphates stimulate the Na+/Pi symporter activities through their SPX domains.